This invention relates to compounds which are inhibitors of elastase, particularly human neutrophil elastase, useful for a variety of physiological and end-use applications. Human neutrophil elastase has been implicated as an agent contributing to the tissue destruction associated with a number of inflammatory diseases such as chronic bronchitis, cystic fibrosis, and rheumatoid arthritis. J. L. Malech and J. I. Gallin, New Engl. J. Med., 317(11), 687 (1987). Elastase possesses a broad range of proteolytic activity against a number of connective tissue macromolecules including elastin, fibronectin, collagen, and proteoglycan. The presence of the enzyme elastase may contribute to the pathology of these diseases.
Normal plasma contains large quantities of protease inhibitors that control a variety of enzymes involved in connective tissue turnover and inflammation. For example, xcex1-1-proteinase inhibitor xcex1-1-PI) is a serine protease inhibitor that blocks the activity of elastase. xcex1-1-PI has received considerable interest because reduction in plasma levels to less than 15% of normal is associated with the early development of emphysema. In addition to plasma derived protease inhibitors, secretory fluids, including bronchial, nasal, cervical mucus, and seminal fluid contain an endogenous protease inhibitor called secretory leukoprotease inhibitor (SLPI) that can inactivate elastase and is believed to play an important role in maintaining the integrity of the epithelium in the presence of inflammatory cell proteases. In certain pathological states xcex1-1-PI and SLPI are inactivated by neutrophil oxidative mechanisms allowing the neutrophil proteases to function in an essentially inhibitor-free environment. For example, bronchial lavage fluids from patients with adult respiratory distress syndrome (ARDS) have been found to contain active elastase and xcex1-1-PI that had been inactivated by oxidation.
In addition to oxidative mechanisms, neutrophils possess non-oxidative mechanisms for eluding inhibition by antiproteases. Neutrophils from patients with chronic granulomatous disease are capable of degrading endothelial cell matrices in the presence of excess xcex1-1-PI. There is considerable in vitro evidence that stimulated neutrophils can tightly bind to their substrates such that serum antiproteases are effectively excluded from the microenvironment of tight cell-substrate contact. The influx of large numbers of neutrophils to an inflammatory site may result in considerable tissue damage due to the proteolysis that occurs in this region.
Applicants have determined that elastase is one of the primary neutrophil proteases responsible for cartilage matrix degeneration as measured by the ability of neutrophil lysate, purified elastase and stimulated neutrophils to degrade cartilage matrix proteoglycan. Furthermore, applicants have previously discovered peptide derivatives useful as elastase inhibitors, exerting valuable pharmacological activities. For example, peptide derivatives useful as elastase inhibitors wherein the terminal carboxyl group has been replaced by a pentafluoroethylcarbonyl (xe2x80x94C(O)C2F5)group and in which the N-terminal amino acid is protected by various heterocycle-containing groups such as a 4-morpholinecarbonyl group are disclosed in European Patent Application OPI No. 0529568, inventors Peet et al., with a publication date of Mar. 3, 1993. Applicants have recently discovered peptidyl elastase inhibitors in which the P2 moiety is substituted with various nitrogen-containing heterocyclic groups.
The present invention relates to compounds having the following formula I 
or a hydrate, isostere, or pharmaceutically acceptable salt thereof wherein
P4 is Ala, bAla, Leu, Ile, Val, Nva, bVal, Nle or a bond;
P3 is Ala, bAla, Leu, Ile, Val, Nva, bVal, Nle or an N-methyl derivative, Pro, Ind, Tic or Tca, or Lys substituted on its epsilon amino group with a morpholino-B-group or Orn substituted on its delta amino group with a morpholino-B-group;
P2 is Pip, Aze, Pro(4-OH), Pro(4-OAc) or Pro(4-OBz1);
R1 is a side chain of Ala, Leu, Ile, Val, Nva or bVal;
X is xe2x80x94CF3, xe2x80x94CF2H, xe2x80x94CFH2, xe2x80x94C(xe2x95x90O)Y, xe2x80x94C(xe2x95x90O)P2xe2x80x2-Y, xe2x80x94CF2C(xe2x95x90O)P2xe2x80x2-Y, xe2x80x94CF2CH(R1xe2x80x2)C(xe2x95x90O)P2xe2x80x2-Y, xe2x80x94CF2CH(R1xe2x80x2)NHC(xe2x95x90O)R3, xe2x80x94CHFCH(R1xe2x80x83)NHC(xe2x95x90O)R3, xe2x80x94H, xe2x80x94C(xe2x95x90O)R3, xe2x80x94CH(R1)C(xe2x95x90O)P2xe2x80x2-Y, xe2x80x94CF2CF3, xe2x80x94CF2(CH2)tCH3, xe2x80x94CF2(CH2)tCOOR4, xe2x80x94CHF(CH2)tCH3, xe2x80x94CF2(CH2)tCONHR4, xe2x80x94CF2(CH2)tCH2OR4, xe2x80x94CF2(CH2)vCHxe2x95x90CH2, xe2x80x94CH2Cl or xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y;
R3 is H, C1-6 alkyl, phenyl, benzyl, cyclohexyl, cyclohexylmethyl;
R4 is H or C1-6 alkyl;
R1xe2x80x2 is a side chain of Ala, Leu, Ile, Val, Nva or bVal;
P2xe2x80x2 is a bond, Ala or Val;
Y is xe2x80x94NHR3, OR3;
t is 2, 3 or 4;
v is 1, 2 or 3;
K is hydrogen, formyl, acetyl, succinyl, benzoyl, t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl, methoxysuccinyl, 1-adamantanesulphonyl, 1-adamantaneacetyl, 2-carboxybenzoyl, phenylacetyl, t-butylacetyl, bis((1-naphthyl)methyl)acetyl, xe2x80x94C(xe2x95x90O)Nxe2x80x94(CH3)2, 
xe2x80x94Axe2x80x94RZ wherein 
RZ is an aryl group containing 6, 10 or 12 carbons suitably substituted by 1 to 3 members selected independently from the group consisting of fluoro, chloro, bromo, iodo, trifluoromethyl, hydroxy, alkyl containing from 1 to 6 carbons, alkoxy containing from 1 to 6 carbons, carboxy, alkylcarbonylamino wherein the alkyl group contains 1 to 6 carbons, 5-tetrazolyl, and acylsulfonamido containing from 1 to 15 carbons, provided that when the acylsulfonamido contains an aryl the aryl may be further substituted by a member selected from fluoro, chloro, bromo, iodo and nitro; 
Z is N or CH, and
B is a group of the formulae 
(the wavy line  being the attachment to the rest of the molecule, i.e., not to Z)
and wherein Rxe2x80x2 is hydrogen or a C1-6 alkyl group; useful as inhibitors of elastase. The compounds of formula I exhibit an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, such as adult respiratory distress syndrome, septicemia, chronic bronchitis, inflammatory bowel disease, disseminated intravascular coagulation, cystic fibrosis, and in the treatment of emphysema.
Isosteres of the compounds of formula I include those wherein (a) one or more of the a-amino residues of the P2-P4 substituents are in its unnatural configuration (when there is a natural configuration) or (b) when the normal peptidic amide linkage [xe2x80x94C(xe2x95x90O)NHxe2x80x94] is modified, such as for example, to form xe2x80x94CH2NHxe2x80x94 (reduced), xe2x80x94COCH2xe2x80x94 (keto), xe2x80x94CH(OH)CH2xe2x80x94(hydroxy), xe2x80x94CH(NH2)CH2xe2x80x94 (amino), xe2x80x94CH2CH2xe2x80x94 (hydrocarbon), xe2x80x94CHxe2x95x90CHxe2x80x94(alkene). Preferably a compound of the invention should not be in an isosteric form; particularly it is preferred that there be no modified peptidic amide group, but if there is, it is preferable to keep the isosteric modifications to a minimum.
A C1-6 alkyl group is taken to include straight, branched, or cyclic alkyl groups, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, sec-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl and cyclopentylmethyl.
The compounds of formua I can form pharmaceutically acceptable salts with any non-toxic, organic or inorganic acid. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono, di and tricarboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxy benzoic., and sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.
Those compounds of formula I wherein X is xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y, can exist in a hydrated or dehydrated form. Hydrates of these triketo compounds of formula I are much more chemically stable than are the dehydrated triketo compounds of formula I wherein X is xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y. For this reason, the hydrates are preferred and any reference in this specification and claims to a triketo compound should be taken to include reference to the corresponding hydrated form as context allows. Moreover, the compounds of this invention are expected to be in the hydrated form under normal physiological conditions.
Each xcex1-amino acid has a characteristic xe2x80x9cR-groupxe2x80x9d, the R-group being the side chain, or residue, attached to the xcex1-carbon atom of the a-amino acid. For example, the R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl. (Thus, throughout this specification, the R1 moiety is the R-group for each indicated xcex1-amino acid). For the specific R-groups or side chains of the xcex1-amino acids reference to A. L. Lehninger""s text on Biochemistry (see particularly Chapter 4) is helpful.
The natural amino acids, with the exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the preferred compounds are the optically active amino acids of the L-configuration; however, applicants contemplate that the amino acids of the formula I compounds can be of either the D- or L-configurations or can be mixtures of the D- and L-isomers, including racemic mixtures. The recognized abbreviations for the xcex1-amino acids are set forth in Table I.
As with any group of structurally related compounds which possesses a particular generic utility, certain groups and configurations are preferred for compounds of formula I in their end-use application.
With respect to the substituent P4, compounds of formula I wherein P4 is Ala or a bond, are preferred. Compounds of formula I wherein P4 is a bond are particularly preferred.
With respect to the substituent P3, compounds of formula I wherein P3 is Ile, Val or Ala, are preferred. Compounds of formula I wherein P3 is Val are particularly preferred.
As for substituent R1, compounds of formula I wherein R1 is xe2x80x94CH(CH3)2 or xe2x80x94CH2CH2CH3, being the characteristic xe2x80x9cR-groupsxe2x80x9d of the amino acids Val and Nva, respectively, are preferred. Compounds of formula I wherein R1 is xe2x80x94CH(CH3)2 are particularly preferred.
With respect to the substiuent X, compounds of formula I wherein X is xe2x80x94CF2CF3, xe2x80x94CF3, xe2x80x94CF2(CH2)tCH3, xe2x80x94CF2(CH2)tCOOR4, xe2x80x94CHF(CH2)tCH3, xe2x80x94CF2(CH2)tCONHR4, xe2x80x94CF2(CH2)tCH2OR4, or xe2x80x94CF2(CH2)vCHxe2x95x90CH2, are preferred. Compounds of formula I wherein X is xe2x80x94CF2CF3 are particularly preferred.
With regard to the substituent K, compounds of formula I wherein K is benzoyl, t-butyloxycarbonyl, carbobenzyloxy, isovaleryl, xe2x80x94C(xe2x95x90O)N(CH3)2, 
Z is N and B is a group of the formulae 
and wherein Rxe2x80x2 is hydrogen or a C1-6 alkyl group are preferred. Compounds of formula I wherein K is 
Z is N and B is a group of the formulae 
and wherein Rxe2x80x2 is hydrogen or a C1-6 alkyl group are particularly preferred.
Specific examples of preferred compounds include:
N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-Nxe2x80x2-[3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-L-2-azetamide;
N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-Nxe2x80x2-[3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-D,L-2-pipecolinamide;
N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-Nxe2x80x2-[3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-trans-4-hydroxyprolinamide;
N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-Nxe2x80x2-[3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl 1-trans-4-acetoxyprolinamide;
N-[4-(4-morpholinylcarbonyl)benzoyl]-L-valyl-Nxe2x80x2-[3,3,4,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl 1-trans-4-benzyloxyprolinamide.
In general, the compounds of formula I may be prepared using standard chemical reaction analogously known in the art and as depicted in Scheme A. 
Scheme A provides a general synthetic scheme for preparing the compounds of formula I.
The P2, P3 and Kxe2x80x94P4 groups can be linked to the free amino group of the amino acid derivative of structure (1). Note that structure (1) represents the P1 moiety wherein the free carboxylic acid group has been substituted with an xe2x80x9cXxe2x80x9d moiety as defined above. The P2, P3 and Kxe2x80x94P4 can be linked to the unproptected, free amino compound (P1-X) by well known peptide coupling techniques. Furthermore, the P1, P2, P3 and Kxe2x80x94P4 groups may be linked together in any order as long as the final compound is Kxe2x80x94P4-P3-P2-P1-X. For example, Kxe2x80x94P4 can be linked to P3 to give Kxe2x80x94P4xe2x80x94P3 which is linked to P2xe2x80x94P1xe2x80x94X; or Kxe2x80x94P4 linked to P3xe2x80x94P2 then linked to an appropriately C-terminal protected Pi and the C-terminal protecting group converted to X.
Generally, peptides are elongated by deprotecting the xcex1-amine of the N-terminal residue and coupling the next suitably N-protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in stepwise fashion, as depicted in Scheme A, or by condensation of fragments (two to several amino acids), or combination of both processes, or by solid phase peptide synthesis according to the method originally described by Merrifield, J. Am. Chem. Soc., 1963, 85, 2149-2154, the disclosure of which is hereby incorporated by reference. When a solid phase synthetic approach is employed, the C-terminal carboxylic acid is attached to an insoluble carrier (usually polystyrene). These insoluble carriers contain a group which will react with the aldehyde group to form a bond which is stable to the elongation conditions but readily cleaved later. Examples of which are: chloro- or bromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many of these resins are commercially available with the desired C-terminal amino acid already incorporated. For compounds of formula I wherein X is H, a linker compound may also be used in the reaction of Scheme A to link a resin to the aldehyde funcationlity of the amino acid derivative of structure (1) wherein X is H. Examples of suitable linker compounds are 
Alternatively, compounds of the invention can be synthesized using automated peptide synthesizing equipment. In addition to the foregoing, peptide synthesis are described in Stewart and Young, xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984); Gross, Meienhofer, Udenfriend, Eds., xe2x80x9cThe Peptides; Analysis, Synthesis, Biologyxe2x80x9d, Vol 1, 2, 3, 5 and 9, Academic Press, New York, 1980-1987; Bodanszky, xe2x80x9cPeptide Chemistry: A Practical Textbookxe2x80x9d, Springer-Verlag, New York (1988); and Bodanszky, et al. xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d Springer-Verlag, New York (1984), the disclosures of which are hereby incorporated by reference.
Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxy-succinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-Cl, or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
The functional groups of the constituent amino acids generally must be protected during the coupling reactions to avoid formation of undesired bonds. The protecting groups that can be used are listed in Greene, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, John Wiley and Sons, New York (1981) and xe2x80x9cThe Peptides: Analysis, Synthesis, Biologyxe2x80x9d, Vol. 3, Academic Press, New York (1981), the disclosure of which is hereby incorporated by reference.
The xcex1-carboxyl group of the C-terminal residue is usually protected by an ester that can be cleaved to give the carboxylic acid. Protecting groups which can be used include: 1) alkyl esters such as methyl and t-butyl, 2) aryl esters such as benzyl and substituted benzyl, or 3) esters which can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters.
The xcex1-amino group of each amino acid to be coupled to the growing peptide chain must be protected. Any protecting group known in the art can be used. Examples of which include: 1) acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonxyls, 1-(p-biphenyl)-1-methylethoxy-carbonyl, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate types such as tertbutyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycaronbyl; 5) alkyl types such as triphenylmethyl and benzyl; 6) trialkylsilane such as trimethylsilane; and 7) thiol containing types such as phenylthiocarbonyl and dithiasuccinoyl. The preferred xcex1-amino protecting group is either Boc or Fmoc, preferably Boc. Many amino acid derivaties suitably protected for peptide synthesis are commercially available.
The xcex1-amino group protecting group of the newly added amino acid residue is cleaved prior to the coupling of the next amino acid. When the Boc group is used, the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane or ethyl acetate. The resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or dimethlformamide. When the Fmoc group is used, the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine or aqueous basic solutions can be used. The deprotection is carried out at a temperature between 0xc2x0 C. and room temperature.
Any of the amino acids bearing side chain functionalities must be protected during the preparation of the peptide using any of the above-described groups. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depends upon the amino acid and presence of other protecting groups in the peptide. The selection of such protecting groups is important in that it must not be removed during the deprotection and coupling of the xcex1-amino group.
For example, when Boc is used as the a-amino protecting group, the following side chain protecting groups are suitable: p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chains of amino acids such as Lys and Arg; p-methylbenzyl, acetamidomethyl, benzyl (Bzl), or t-butylsulfonyl moieties can be used to protect the sulfide containing side chains of amino acids such as cysteine and benzyl (Bzl) ether can be used to protect the hydroxy containing side chains of amino acids such as Ser or Thr.
When Fmoc is chosen for the xcex1-amine protection, usually tert-butyl based protecting groups are acceptable. For instance, Boc can be used for lysine, tert-butyl ether for serine and threonine and tert-butyl ester for glutamic acid.
Once the elongation of the peptide is completed all of the protecting groups are removed. When a liquid phase synthesis is used, the protecting groups are removed in whatever manner is dictated by the choice of protecting groups. These procedures are well known to those skilled in the art.
When a solid phase synthesis is used, the peptide is cleaved from the resin usually simultaneously with the protecting group removal. When the Boc protection scheme is used in the synthesis, treatment with anhydrous HF containing additivies such as dimethyl sulfide, anisole, thioanisole, or p-cresol at 0xc2x0 C. is the preferred method for cleaving the peptide from the resin. The cleavage of the peptide can also be accomplished by other acid reagents such as trifluoromethanesulfonic acid/trifluoroacetic acid mixtures. If the Fmoc protection scheme is used the N-terminal Fmoc group is cleaved with reagents described earlier. The other protecting groups and the peptide are cleaved from the resin using solution of trifluoroacetic acid and various additives such as anisole, etc.
For those compounds of formula I wherein X is H, the peptide compound of formula I may be cleaved from the linker compound and resin with aqueous acid/formaldehyde.
Alternatively, the compounds of formula I may be prepared using standard chemical reaction analogously known in the art and as depicted in Scheme B 
Scheme B provides an alternative general synthetic scheme for preparing the compounds of formula I.
The P2, P3 and Kxe2x80x94P4 groups can be linked to the free amino group of the amino alcohol derivative of structure (2) as described previously in Scheme A to give the peptido alcohol of structure (3).
The alcohol functionality of the peptido alcohol of structure (3) is then oxidized by techniques and procedures wellknown and appreciated by one of ordinary skill in the art, such as a Swern Oxidation using oxalyl chloride and dimethylsulfoxide, to give the compounds of formula I.
Starting materials for use in Schemes A and B are readily available to one of ordinary skill in the art. For example, amino acids P2, P3 and Kxe2x80x94P4 wherein K is hydrogen are commercially available and the linker compound of structure (L1) is described in J. Am. Chem. Soc., 114, 3157-59 (1992). In addition, substituted amino acids Kxe2x80x94P4 wherein
K is acetyl, succinyl, benzoyl, t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl, methoxysuccinyl, 1-adamantanesulphonyl, 1-sdamantaneacetyl, 2-carboxybenzoyl, phenylacetyl, t-butylacetyl, bis [(1-naphthyl)-methyl]acetyl or xe2x80x94Axe2x80x94RZ wherein
A is 
Rz is an aryl group containing 6, 10 or 12 carbons suitably suitably substituted by 1 to 3 members selected independently from the group consisting of fluoro, chloro, bromo, iodo, trifluoromethyl, hydroxy, alkyl containing from 1 to 6 carbons, alkoxy containing from 1 to 6 carbons, carboxy, alkylcarbonylamino wherein the alkyl group contains 1 to 6 carbons, 5-tetrazolyl, and acylsulfonamido (i.e., acylaminosulfonyl and sulfonylaminocarbonyl) containing from 1 to 15 carbons, provided that when the acylsulfonamido contains an aryl the aryl may be further substituted by a member selected from fluoro, chloro, bromo, iodo and nitro; and such other terminal amino protecting groups which are functionally equivalent thereto are described in European Patent Application OPI No. 0363284, Apr. 11, 1990.
Starting amino compounds of structure (1) are readily available to one of ordinary skill in the art. For example, certain protected amino compounds of structure (1) wherein X is H are well known in the literature and are also described in European Patent Application OPI No. 0275101, Jul. 20, 1988 and in European Patent Application OPI No. 0363284, Apr. 11, 1990. In addition, amino compounds of structure (1) wherein X is xe2x80x94CF3, xe2x80x94CF2(CH2)tCH3, xe2x80x94CF2(CH2)tCOOR4, xe2x80x94CF2(CH2)tCONHR4, xe2x80x94CF2(CH2)tCH2OR4, xe2x80x94CF2CF3, and xe2x80x94CF2(CH2)tCHxe2x95x90CH2 are described in European Patent Application OPI No. 0503203, Sep. 16, 1992. Amino compounds of structure (1) wherein X is xe2x80x94CF3, xe2x80x94CF2H, xe2x80x94C(xe2x95x90O)xe2x80x94Y, xe2x80x94CF2CH(R1xe2x80x2)xe2x80x94C(xe2x95x90O)P2xe2x80x2xe2x80x94Y and xe2x80x94C(xe2x95x90O)xe2x80x94P2xe2x80x2xe2x80x94Y are described in European Patent Application OPI No. 0195212, Sep. 24, 1986. Amino compounds of structure (1) wherein X is xe2x80x94CF2CH(R1xe2x80x2)NHC(xe2x95x90O)R3 are described in OPI No. 0275101, Jul. 20, 1988 and amino comounds of structure (1) wherein X is xe2x80x94CHFCH(R1xe2x80x2)NHC(xe2x95x90O)R3 may be prepared by analogous procedures using bromo-fluroacetic acid, ethyl ester in place of bromo-difluroacetic acid, ethyl ester. Amino compound of structure (1) wherein X is xe2x80x94CFH2 are described in Biochem. J. (1987), 241, 871-5, Biochem. J. (1986), 239, 633-40 and U.S. Pat. No. 4,518,528, May 21, 1985. Amino compounds of structure (1) wherein X is xe2x80x94CO2R3, C(xe2x95x90O)xe2x80x94R3 and xe2x80x94CH(R1)xe2x80x94C(xe2x95x90O)P2xe2x80x2xe2x80x94Y are described in European Patent Application OPI No. 0363284, Apr. 11, 1990. Amino compounds of structure (1) wherein X is xe2x80x94CF2CH(R1xe2x80x2)NHC(xe2x95x90O)xe2x80x94R3 are described in European Patent Application OPI No. 0275101, Jul. 20, 1988. In addition, amino compounds of structure (15) wherein X is xe2x80x94CF2CF3 are described in European Patent Application OPI No. 0410411, Jan. 30, 1991. The linker compound trans-4-(aminomethyl)-cyclohexanecarboxylic acid, benzyl ester used in synthesis of compounds of formula I wherein X is H is prepared from the corresponding acid as described in J. Am. Chem. Soc. 1992, 114, 3156-3157.
In addition, other starting materials for use in Schemes A and B may be prepared by the following synthetic procedures which are well known and appreciated by one of ordinary skill in the art.
Substituted amino acids Kxe2x80x94P4 of structure wherein K is 
Z is N or CH, and
B is a group of the formulae 
wherein Rxe2x80x2 is hydrogen or a C1-6 alkyl group are prepared using standard chemical reactions analogously known in the art.
The procedure for preparing the substituted amino acids Kxe2x80x94P4 wherein K is 
B is a xe2x80x94C(xe2x95x90O)xe2x80x94 is outlined in Scheme C wherein P4 and Z are as previously defined or are the functional equivalents of these groups. 
Specifically the amino acids Kxe2x80x94P4 wherein K is 
B is a xe2x80x94C(xe2x95x90O)xe2x80x94 are prepared by coupling of the amino acid Kxe2x80x94P4 wherein K is hydrogen with an acid chloride of structure (4) in the presence of from one to four molar equivalents of a suitable amine which can act as a hydrogen halide acceptor. Suitable amines for use as hydrogen halide acceptors are tertiary organic amines such as tri-(lower alkyl)amines, for example, triethylamine, or aromatic amines such as picolines, collidines, and pyridine. When pyridines, picolines, or collidines are employed, they can be used in high excess and act therefore also as the reaction solvent. Particularly suitable for the reaction is N-methylmorpholine (xe2x80x9cNMMxe2x80x9d). The coupling reaction can be performed by adding an excess, such as from 1-5, preferably about a 4-fold molar excess of the amine and then the acid chloride of structure (4), to a solution of the amino acid Kxe2x80x94P4 wherein K is hydrogen. The solvent can be any suitable solvent, for example, petroleum ethers, a chlorinated hydrocarbon such as carbon tetrachloride, ethylene chloride, methylene chloride, or chloroform; a chlorinated aromatic such as 1,2,4-trichlorobenzene, or o-dichlorobenzene; carbon disulfide; an ethereal solvent such as diethylether, tetrahydrofuran, or 1,4-dioxane, or an aromatic solvent such as benzene, toluene, or xylene. Methylene chloride is the preferred solvent for this coupling reaction. The reaction is allowed to proceed for from about 15 minutes to about 6 hours, depending on the reactants, the solvent, the concentrations, and other factors, such as the temperature which can be from about 0xc2x0 C. to about 60xc2x0 C., conveniently at about room temperature, i.e. 25xc2x0 C. The N-protected amino acids Kxe2x80x94P4 wherein K is 
B is a xe2x80x94C(xe2x95x90O)xe2x80x94 can be isolated from the reaction mixture by any appropriate techniques such as by chromatography on silica gel.
The substituted amino acids Kxe2x80x94P4 wherein K is 
B is other than a xe2x80x94C(xe2x95x90O)xe2x80x94 can be prepared analogously, merely by substituting the appropriate intermediate 
B is other than a xe2x80x94C(xe2x95x90O)xe2x80x94 and A is Cl or OH (the corresponding acid, acid chloride or sulphonyl chloride) for the compound of structure (5) in Scheme C.
The acid chloride of structure (4) and the appropriate intermediate of formula 
B is other than a xe2x80x94C(xe2x95x90O)xe2x80x94 and A is Cl or OH (the corresponding acid, acid chloride or sulphonyl chloride) are commercially available or may be readily prepared by techniques and procedures well known and appreciated by one of ordinary skill in the art.
For example, the appropriate intermediates of formula 
may be prepared as outlined in Scheme D wherein all substituents are as previously defined. 
Scheme D provides a general synthetic procedure for preparing the appropriate intermediates of formula 
Z is as previously defined.
In step a, the carboxylic acid functionality of the appropriate 2,5-pyridinedicarboxylic acid, 2-methyl ester (6) (Nippon Kagaku Zasshi, 1967, 88, 563) is converted to its acid chloride using techniques and procedures well known and appreciated by one of ordinary skill in the art, such as thionyl chloride, to give the corresponding 6-carbomethoxynicotinoyl chloride (7).
In step b, the acid chloride (7) is amidated with morpholine (8) by techniques and procedures well known and appreciated by one of ordinary skill in the art to give the corresponding 5-(morpholine-4-carbonyl)-2-pyridinecarboxylic acid, methyl ester (9).
In step c, the methyl ester functionality (9) is hydrolyzed by techniques, and procedures well known and appreciated by one of ordinary skill in the art, with for example, lithium hydroxide in methanol, to give 5-(morpholine-4-carbonyl)-2-pyridine carboxylic acid (10).
In addition, the appropriate intermediate of formula 
may be prepared as outlined in Scheme E wherein all substituents are as previously defined. 
Scheme E provides a general synthetic procedure for preparing the appropriate intermediates of formula 
Z is as previously defined.
In step a, the free carboxylic acid functionality of 2,5-pyridinedicarboxylic acid, 2-methyl ester (6) (Nippon Kagaku Zasshi, 1967, 88, 563) is converted to its t-butyl ester using techniques and procedures well known and appreciated by one of ordinary skill in the art, such as the t-butyl alcohol adduct of dicyclohexylcarbodiimide (Synthesis, 1979, 570), to give the corresponding 2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-t-butyl ester (11).
For example, the 2,5-pyridinedicarboxylic acid, 2-methyl ester (6) is combined with a molar excess of the t-butyl alcohol adduct of dicyclohexylcarbodiimide in an appropriate organic solvent, such as methylene chloride. The reaction is typically conducted at a temperature range of from 0xc2x0 C. to room temperature and for a period of time ranging from 2-24 hours. The 2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-t-butyl ester (11) is isolated from the reaction mixture by standard extractive methods as is known in the art and may be purified by crystallization.
In Step b, the methyl ester functionality of (11) is amidated with morpholine (8) to give the corresponding 6-(morpholine-4-carbonyl)nicotinic acid, t-butyl ester (12). For example, the 2,5-pyridinedicarboxylic acid, 2-methyl ester, 5-t-butyl ester (11) is contacted with a molar excess of morpholine in an appropriate organic solvent, such as tetrahydrofuran. The reaction is typically conducted at a temperature range of from room temperature to reflux and for a period of time ranging from 5 hours to 3 days. The 6-(morpholine-4-carbonyl)nicotinic acid, t-butyl ester (12) is isolated from the reaction mixture by standard extractive methods as is known in the art and may be purified by crystallization.
In step c, the t-butyl ester functionality of (12) is hydrolyzed, with for example, HCl in nitromethane, to give the corresponding, 6-(morpholine-4-carbonyl)nicotinic acid (13).
Amino compounds of structure (1) wherein X is xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y may be prepared by techniques and procedures well known by one of ordinary skill in the art.
For example, amino compounds of structure (1) wherein X is xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y may be prepared as described in Scheme F wherein all substituents are as previously defined. 
Specificially, the amino compounds of structure (1) wherein X is xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94Y can be prepared, as illustrated in scheme F, by treatment of the appropriate N-Boc protected tricarbonyl compound (20) with an appropriate acid, such as hydrogen chloride in ethyl acetate or nitromethane or trifluoroacetic acid neat or as a solution in methylene chloride, followed by generation of the free base (21) using an appropriate base.
Intermediate (20) is generated from ylide (19) by treatment with (a) ozone and dimethyl sulfide or (b) singlet oxygen or (c) Oxone(copyright). The ozonolysis reaction can be conveniently performed by, for example, bubbling an excess of ozone through a cooled solution of the appropriate ylide of structure (20). Suitable solvents include any nonreactive solvent in which the ylide of structure (20) is soluble, for example, alkyl esters of simple alkanoic acids such as ethyl acetate; the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane, and methylene chloride; the aromatic hydrocarbons such as benzene, toluene, and xylene; a chlorinated aromatic such as 1,2,4-trichlorobenzene and o-diclorobenzne; or an ethereal solvent such as diethyl ether, tetrahydrofuran (THF), and 1,4-dioxane. Methylene chloride is preferred.
The temperature of the ozonolysis reaction mixture can be any temperature conductive to the reaction, typically from about xe2x88x9278xc2x0 C. to about 0xc2x0 C. preferably from about xe2x88x9278xc2x0 C. to about xe2x88x9235xc2x0 C., and most preferably about xe2x88x9270xc2x0 C. The time of the reaction will vary depending on the ylide, the conentration of the reactants, the temperature and other factors. Conveniently, ozone is bubbled into the reaction mixture until the solution turns blue indicating an excess of ozone.
The ozonide is then treated with an excess of a reducing agent such as zinc metal or preferably dimethylsulfide. Compound (20) is isolated as the hydrate from the reaction mixture in any convenient manner, typically by solvent removal (via evaporation). Purification may be accomplished by, for example, flash chromatography (Still, W. C.; Kahn, M.; Mitra, A.; J. Org. Chem. 1978, 43, 2923).
Oxone(copyright) may be used in place of ozone whe a milder and more selective reagent is desired. Typically, ylide (19) is treated with 1.5 equivalents of Oxone(copyright) in THFxe2x80x94H2O and the resultant hydrated tricarbonyl is isolated from the reaction mixture.
Oxidations utilizing singlet oxygen are well known. More specifically, singlet oxygen oxidation of an ylide to produce a tricarbonyl ester has been reported by H. Wasserman et al., J. Amer. Chem. Soc., 11, 371 (1989). Singlet oxygen can be generated by dye-sensitized excitation of oxygen. Suitable dyes include Rose Bengal, Eosin Y and methylene blue. Other sensitizers include inaphthalenethiophene. Typically, Rose Bengal and Eosin Y are attached to a basic anion-exchange resin and methylene blue is attached to an acidic cation-exchange resin. Excitation is accomplished with a UV lamp such as a tungsten-iodine lamp. Suitable solvents are any solvents which promote and do not interfere with the desired reaction. Such solvents include the aromatic hydrocarbons such as benzene and toluene; hydrocarbons such as hexane; ethereal solvents such as diethyl ether, tetrahydrofuran (THF), 1,4-dioxane; chlorinated hydrocarbons such as dichloromethane and chloroform; and carbon disulfide. Mixtures are operable. The temperature of the reaction mixture can be any suitable temperature from about xe2x88x9278xc2x0 C. to about 30xc2x0 C. typically from about xe2x88x9278xc2x0 C. to xe2x88x9250xc2x0 C. The time of the reaction will vary depending on the reactant, the solvent, concentrations, and temperature and can be from about 1 minute to about 2 hours. Purification and isolation can by by those methods desrbied above for specification and isolation of product from ozonolysis reaction mixture.
The N-Boc protected ylide of structure (19) is prepared by coupling of the N-Boc protected amino acid of structure (14) with the phosphonium ylide of structure (16) using a water-soluble carbodiimide (WSCDI) such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (18) in the presence of 4-dimethylaminopyridine (DMAP) in a suitable solvent such as THF or dichloromethane. The reaction will require from about 30 minutes to about 12 hours, typically about 2 to 3 hours, depending on the amino acid, the ylide, the solvent(s), and the temperature which can be from about xe2x88x9215xc2x0 C. to about 60xc2x0 C., but typically at 0xc2x0 C. Isolation and purification is accomplished by filtering the reaction mixture to remove solid products and subseqently chromatographing the filtrate, for example, on silica gel.
The phosphorous ylide, Wittig reagent, of structure (16) is prepared from the corresponding xcex1-halocarboxylic acid derivative of structure (15) in the usual manner, that is, by reacting the xcex1-halo ester with a tertiary phosphine such as triphenylphosphine to yield a phosphonium salt. When treated with a strong base such as an organolithium compound, for example, lithium diisopropylamide (LDA), sodium hydride, or sodium amide, the acidic proton is removed and the desired ylide is formed. Suitable solvents used in forming the Wittig reagent include any nonreactive solvent, for example, the aromatic hydrocarbons such as benzene or toluene, the chlorinated hydrocarbons such as carbon tetrachloride, chloroform, or methylene chloride, or the ethereal solvents such as diethyl ether or THF.
The reaction can conveniently be performed at from about 0xc2x0 C. to about 60xc2x0 C., typically at room temperature, that is about 25xc2x0 C. The halo group of the a-halo ester is preferably a bromo group, but can be a chloro or iodo group or can by any good leaving group which forms a stable phosphonium salt such as a mesylate or tosylate group.
In addition, amino compounds of structure (1) wherein X is xe2x80x94CHF(CH2)tCH3 may be prepared as described in Scheme G wherein all substituents are as previously defined 
Scheme G provides a general synthetic procedure for preparing the amino compounds of structure (1) wherein X is xe2x80x94CHF(CH2)tCH3. In Scheme G, all substituents are as previously defined unless otherwise indicated.
In step a, the appropriate acid of structure (22) is amidated with N-methyl-N-methoxyamine by techniques and procedures well known and appreciated by one of ordinary skill in the art, such as a coupling reaction using 1,3-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) to give the corresponding amide of structure (23).
In step b, the appropriate amide of structure (23) is alkylated with the appropriate alkyl metal compound of structure (24) to give the corresponding keto compound of structure (25).
For example, the appropriate amide of structure (23) is treated with the alkyl metal compound of structure (24) in a suitable aprotic, anhydrous organic solvent wuch as tetrahydrofuran or diethyl ether. The reaction is typically conducted at a temperature range of from xe2x88x9278xc2x0 C. to xe2x88x9240xc2x0 C. and for a period of time ranging from 30 minutes to 5 hours. The corresponding keto compound of structure (25) is recovered from the reaction zone by extractive methods as is known in the art and may be purified by chromatography.
In step c, the appropriate keto compound of structure (25) is fluorinated with the N-fluorosulfonimide compound of structure (26), or the alternative fluorination reagents (27), (28) or (29) to give the protected amino compounds of structure (30) which is the amino compound of structure (1) in which the amino terminal group is substituted with a Boc group and X is xe2x80x94CHF(CH2)tCH3.
For example, the appropriate keto compound of structure (25) is treated with an appropriate non-nucleophilic base, such as lithium diisopropylamide in a suitable anhydrous aprotic organic solvent, such as tetrahydrofuran at a temperature range of from xe2x88x9278xc2x0 C. to xe2x88x9240xc2x0 C. and for a period of time ranging from 5 minutes to 2 hours. The reaction mixture is then treated with the N-fluorosulfonimide compound of structure (25) and the reaction conducted at a temperature range of from xe2x88x9278xc2x0 C. to xe2x88x9240xc2x0 C. and for a period of time ranging from 30 minutes to 10 hours. The N-t-Boc protected amino compounds of structure (1) wherein X is xe2x80x94CHF(CH2)tCH3 is recovered from the reaction zone by extractive methods as is known in the art and may be purified by chromatography.
Alternate routes for the preparation of compounds of structure (1) wherein Xxe2x95x90xe2x80x94CF2CF3, is shown in scheme H.
The required starting material defined by compound (31) is readily available either commercially or by applying known prior art principles and techniques. The term xe2x80x9cPgxe2x80x9d refers to a suitable protecting group as more fully defined previously.
In Scheme H, step a the protected amino acid (31) is transformed into the hydroxamate (32). This amidation can be performed utilizing a coupling reaction as between two amino acids using the protected amino acid (31) and the N-alkyl O-alkylhydroxylamine. The standard coupling reaction can be carried out using standard coupling procedures as described previously for the coupling between two amino acids to provide the hydroxamate (32).
In step b, the protected hydroxamate (32) is transformed into the protected pentafluoroketone (34) (or (35)]. This reaction can be performed utilizing a reaction of the type described in the following reference M. R. Angelastro, J. P 
Burkhart, P. Bey, N. P. Peet, Tetrahedron Letters, 33 (1992), 3265-3268.
In step c, the hydroxamate (32) is deprotected under conditions well known in the art as described by T. H. Green xe2x80x9cProtection Groups in Organic Synthesisxe2x80x9d, John Wiley and Sons, 1981, Chapter 7, to provide the deprotected hydroxamate. The deprotected hydroxamate is elongated by coupling the next suitably protected amino acid through a peptide linkage using the methods previously described, or by condensation of fragments, or combination of both processes to provide the elongated peptide (33).
In step d, the ketone (34) is deprotected under conditions as previously described. The deprotected ketone (34) is elongated by coupling the next suitably protected amino acid through a peptide linkage using the methods previously described, or by condensation of fragments, or combination of both processes to provide the elongated ketone (35).
Alternatively, the corresponding N-protected amino acid ester of (31) [i.e. PgNHxe2x80x94CH(R1)C(xe2x95x90O)OR2, (32a), wherein R2 and Pg are as defined above] can be substituted for the hydroxamate (32). The corresponding protected amino acid esters of (31) are commercially available or easily synthesized from (31) by procedures well known by one of ordinary skill in the art. In step b, the amino acid ester (32a), is transformed into the N-protected pentafluoroketone (34) (or (35)] in a manner directly analogous to that used for the corresponding hydroxamate. Steps c and d would be the same as those employed when utilizing the hydroxamate (32).
For example, the amino acid ester (32a) may be reacted with a suitable perfluorinating agent, such as, from 4-8 equivalents of perfluoroethyl iodide or perfluoroethyl bromide. Said reaction is carried out in the presence of a suitable alkali metal base, for example from 4-8 equivalents of MeLi/LiBr in an appropriate anhydrous solvent (or mixed solvents), such as ether, THF, or toluene. Other examples of suitable alkali metal bases include t-BuLi, EtMgBr, PhMgBr, n-BuLi, and the like. The reaction is carried out at reduced temperature of from xe2x88x92100xc2x0 C. to 0xc2x0 C., preferably from xe2x88x9230xc2x0 C. to xe2x88x9280xc2x0 C., to provide the protected perfluoropropyl amino ketone and the protected perfluorobutyl amino ketone, respectively. Steps c and d would be the same as those employed when utilizing the hydroxamate (32).
Alternatively, the N-protected amino acid ester (32a) could first be deprotected and coupled with a suitably N-protected peptide in the presence of a suitable coupling agent and in the presence of an appropriate coupling solvent. The subsequently formed N-protected peptide ester [KP4P3P2NHxe2x80x94CH(R1)C(xe2x95x90O)OR2, (33a)] would then be per-fluorinated in a manner directly analogous to that used for the corresponding hydroxamate. Steps c and d would be the same as those employed when utilizing the hydroxamate (33).
All of the amino acids employed in the synthesis of Formula I are either commercially available or are easily synthesized by one skilled in the pertinent art. For example, the amino acid derivative Pro(4-Ac) defined in P2 can be made by esterifying a Pro residue by utilizing techniques well-known by one of ordinary skill in the art.
The following examples present typical syntheses as described in Schemes A through H. These examples are understood to be illustrative only and are not intended to limit the scope of the present invention in any way. As used herein, the following terms have the indicated meanings: xe2x80x9cgxe2x80x9d refers to grams; xe2x80x9cmmolxe2x80x9d refers to millimoles; xe2x80x9cmLxe2x80x9d refers to milliliters; xe2x80x9cbpxe2x80x9d refers to boiling point; xe2x80x9cxc2x0 C.xe2x80x9d refers to degrees Celsius; xe2x80x9cmm Hgxe2x80x9d refers to millimeters of mercury; xe2x80x9cxcexcLxe2x80x9d refers to microliters; xe2x80x9cxcexcgxe2x80x9d refers to micrograms; and xe2x80x9cxcexcMxe2x80x9d refers to micromolar; xe2x80x9cDMExe2x80x9d refers to 1,2-dimethoxyethane; xe2x80x9cDCCxe2x80x9d refers to dicyclohexylcarbodiimide; xe2x80x9chxe2x80x9d refers to hour; xe2x80x9cDMFxe2x80x9d refers to N,Nxe2x80x2-dimethylformamide; xe2x80x9cconcxe2x80x9d refers to concentrated; xe2x80x9cNMMxe2x80x9d refers to N-methylmorpholine, xe2x80x9cin vacuoxe2x80x9d refers to removal of solvent under reduced pressure.